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Juvenile direct-care officers working in juvenile correctional facilities historically responded to critical and potentially aggressive incidents using nontherapeutic strategies. The purpose of this study was to examine and to understand the lived experiences of direct-care officers' use of de-escalation skills for managing violent and disruptive behaviors in juvenile correctional facilities. The findings from this study may add to the existing literature by describing juvenile correctional officers' experiences as well as to better understand their perceptions and attitude using de-escalation strategies in correctional environments. The social learning and self-efficacy theories provided the conceptual framework to examine and understand their experiences. The phenomenological design was used to examine the lived experiences of 9 juvenile direct-care officers use of de-escalation strategies to respond to disruptive and aggressive incidents within the juvenile correctional facility. The 9 direct-care officers participated in audio-recorded interviews that were transcribed and analyzed using Moustakas's phenomenological steps that identified 3 themes using de-escalation strategies: to avoid use of force and reduce liability of injuries; to resolve conflicts using their words to de-escalate the youth or the situation; and to use according to their training, perceived level of confidence, and effectiveness of de-escalation strategies. Understanding direct-care officers' perceptions of use of de-escalation may result in positive social change for fostering caring and safe living correctional environments and strengthen current training curriculums for working with aggressive and disruptive behaviors.

Les glycosyltransférases (GTs) sont une famille importante d’enzymes responsable de la biosynthèse des chaînes de glycosaminoglycane (GAG) des protéoglycanes, composants clés de la matrice extracellulaire et de la membrane plasmique cellulaire impliqués dans la communication, l'adhésion, la migration et la prolifération cellulaires. Les GTs jouent donc un rôle central dans de nombreux processus physiologiques et physiopathologiques tels que les cancers ou encore les maladies dégénératives et génétiques. Parmi ces GTs, la ß1,4-galactosyltransférase 7 (ß4GalT7) est une cible thérapeutique potentielle puisqu'elle : i) catalyse une étape précoce et majeure de la biosynthèse des chaînes de GAG, ii) est impliquée dans une forme rare de maladie génétique des tissus conjonctifs, le syndrome d’Ehlers-Danlos, iii) prend en charge des xylosides exogènes modulant son activité in vitro et in vivo. Ce travail de thèse s'organise sur une étude structure/fonction de cette enzyme afin de cerner les résidus d'acides aminés clés dans l'interaction de l'enzyme avec un substrat modèle, le 4-methylombelliferyl-ß-D-xylose (4-MOX). Les résidus Y194, Y196 et Y199 ont ainsi été identifiés comme clés dans l'architecture du site de fixation du substrat accepteur et dans l'interaction de l'enzyme avec le xyloside. Au contraire, les résidus H195, R226 et le résidu R270, muté dans la forme progéroïde du syndrome d'Ehlers-Danlos, apparaissent comme des résidus « modulant » l'activité de l'enzyme, notamment du fait d'interactions moléculaires impliquant leur squelette peptidique pour H195 et R226 et une boucle flexible pour R270. Ces travaux ont permis de guider la synthèse d’analogues xylosidiques visant à inhiber l'activité de la ß4GalT7 humaine. Parmi les propositions, un dérivé fluoré du 4-MOX apparaît comme un inhibiteur efficace de la ß4GalT7 in vitro et in cellulo. Faiblement cytotoxique, ce dérivé réduit la prolifération des cellules de lignées cancéreuses SW1353 et MB MDA 231. Ces résultats ouvrent la perspective de nouvelles stratégies thérapeutiques utilisant les xylosides comme agents potentiels dans le traitement de cancers ou encore des maladies génétiques des tissus conjonctifs / Glycosyltransferases (GTs) are an important family of enzymes involved in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans which are key components of cell plasma membranes and of the extracellular matrix, and are thus implicated in cell communication, adhesion, migration and proliferation. GTs are thus key players in numerous pathophysiological processes such as cancers, degenerative and genetic diseases. Among these GTs, ß1,4-galactosyltransférase 7 (ß4GalT7) is a potential therapeutic target since : i) it catalyzes a rate-limiting step in the early phase of the GAG chains biosynthesis, ii) it is implicated in a rare genetic connective tissue disorder (Ehlers-Danlos Syndrome), iii) its in vitro and in vivo activity can be modulated by exogenous xyloside molecules. This PhD work is focused on a structure/function study of the enzyme aiming to identify key amino acid residues that interact with 4-methylumbelliferyl-ß-D-xylose (4-MUX), taken as reference substrate. Y194, Y196 and Y199 have been identified as key residues for the architecture of the acceptor substrate binding site and establish interactions with 4-MUX. By contrast, H195, R226 and R270, a residue mutated in the progeroid form of Ehlers-Danlos Syndrome, should rather be considered as “modulating” residues towards the ß4GalT7 activity, H195 and R226 interacting with 4-MUX with their polypeptide backbone, and R270 via a flexible loop. This work guided the design of xylosidic compounds that would potentially inhibit the ß4GalT7 activity. Thus, a fluorinated derivative of 4-MUX appeared as an efficient in vitro and in cellulo inhibitor of the enzyme. Poorly cytotoxic, this compound also reduced the proliferation rate of cancer cells SW1353 and MB MDA 231. Altogether, these results offer new therapeutic strategies using xylosides as potential therapeutic agents in the treatment of cancer or rare genetic disorders

Les cellules β pancréatiques synthétisent et sécrètent l'insuline, seule hormone hypoglycémiante de l'organisme. Dans le cas du diabète de type 2, du diabète de type 1 et suite à une greffe d'îlots de Langherans, on observe une diminution drastique de cette masse fonctionnelle β. L'hyperglycémie chronique et la libération de cytokines proinflammatoires jouent un rôle cytotoxique prépondérant dans ces phénomènes. Dans le but de préserver ou de restaurer cette masse fonctionnelle β chez les patients diabétiques, notre objectif était d'identifier des outils permettant de protéger des effets délétères de l'hyperglycémie chronique et des cytokines proinflammatoires, en s'intéressant à 3 cibles potentielles. Nous montrons tout d'abord que les activités du système ubiquitine protéasome (UPS), impliqué dans la dégradation de protéines, sont altérées en condition d'hyperglycémie chronique. Ces altérations sont corrélées à l'émergence d'un programme apoptotique au sein des cellules β. L'activation du récepteur du GLP-1 (Glucagon-Like Peptide-1), stratégie thérapeutique majeure dans le diabète de type 2, protège l'UPS des effets délétères de l'hyperglycémie chronique. Le facteur de transcription CREB (cAMP Response Element Binding Protein), essentiel pour la survie et la fonction des cellules β, est dégradé par l'hyperglycémie chronique et l'inflammation. Nous montrons que la prévention de sa dégradation prévient les effets de l'hyperglycémie chronique, mais pas de l'inflammation. Ces observations nous ont amenés à étudier la MAP3 kinase Tpl2 (Tumor progression locus 2), impliquée, notamment via l'activation de ERK1/2 (Extra-cellular Regulated Kinases 1/2), dans les processus inflammatoires d'autres types cellulaires. Nous montrons que Tpl2 est exprimé dans la lignée cellulaire β INS-1E, et dans les îlots murins et humains, et qu'elle gouverne spécifiquement l'activation des kinases ERK1/2 induite par les cytokines proinflammatoires IL-1β, TNFα et IFNγ. Cette protéine est surexprimée dans des conditions d'inflammation (in vitro et modèle de diabète murin). L'inhibition de Tpl2 protège contre l'apoptose induite par les cytokines, dans les INS-1E et les îlots de souris et restaure la capacité sécrétrice d'insuline des ilots de souris altérée suite à une exposition aux cytokines. En combinaison avec un analogue du GLP-1, l'inhibition pharmacologique de cette kinase protège totalement contre les effets délétères des cytokines sur la fonction et la survie des îlots humains. Ces données suggèrent que l'inhibition pharmacologique de la kinase Tpl2, seule ou en combinaison avec un analogue du GLP-1, pourrait constituer de nouvelles stratégies thérapeutiques pour protéger contre l'altération de la masse fonctionnelle β pouvant survenir chez des patients diabétiques de type 2 ou après la transplantation d'îlots. / Pancreatic β cells synthesize and secrete insulin, the sole hormone of the organism able to reduce glycemia. In the course of type 2 and type 1 diabetes, and after islet transplantation, there is a drastic loss of function and mass of these cells. Among the common origins of this decrease, chronic hyperglycemia and the release of proinflammatory cytokines play major roles. With the aim to preserve or to restore this functional β cell mass in diabetic patients, our objective was to identify tools able to protect against deleterious effects of these two phenomenons, interesting in three potential targets. We first demonstrated that the ubiquitin-proteasome system (UPS) activities, that degrade proteins, are altered in β cells exposed to chronic hyperglycemia, and correlated with apoptosis. Activation of the GLP-1 (Glucagon-Like Peptide-1) receptor, a key therapeutic strategy in type 2 diabetes, protects UPS from deleterious effects of chronic hyperglycemia. The transcription factor CREB (cAMP Response Element Binding Protein), crucial for β cell survival and function, is involved in deleterious effects of chronic hyperglycemia and inflammation. We demonstrated that prevention of CREB degradation protects β cells from chronic hyperglycemia, but not from the deleterious effects of the proinflammatory cytokines. These observations prompted us to study the MAP3 kinase Tpl2 (Tumor progression locus 2), known to be implicated in inflammatory process in other cell types, through the activation of the kinases ERK1/2 (Extra-cellular Regulated Kinases 1/2). We showed that Tpl2 is expressed in INS-1E clonal β cells and in mouse and human islets, and that it governs specifically the activation of ERK1/2 in response to proinflammatory cytokines IL-1β, TNFα and IFNγ. This protein is overexpressed by inflammatory conditions and in a rat type 2 diabetes model. Inhibition of Tpl2 protects against cytokine-induced apoptosis in INS-1E and in mouse islets. Furthermore, the capacity of mouse islets to secrete insulin in response to glucose, that is altered by a chronic exposure to cytokines, is restored by Tpl2 inhibitor. Finally, we showed that in combination with GLP-1 analog (Exendin-4), Tpl2 inhibitor can entirely restore the survival and function in human islets cultured in pro-inflammatory conditions. These results suggest that pharmacological inhibition of Tpl2, alone or in combination with Exendin-4, may be novel therapeutic strategies to alleviate β-cell failure observed in Type 2 diabetes and islets transplantation.

La tularémie est une zoonose liée à la bactérie Francisella tularensis, hautement pathogène pour l’homme. La sous espèce la plus virulente, F. tularensis subsp. tularensis, est retrouvée uniquement en Amérique du Nord, alors que la sous-espèce F. tularensis subsp. holarctica est présente dans tout l’hémisphère Nord. En France toutes les souches appartiennent au biovar I de la sous-espèce holarctica et plus précisément au groupe phylogénétique B.FTNF002-00. Bien que rarement grave en France, la tularémie pose le problème de taux d’échecs thérapeutiques élevés, jusqu’à 25% en cas de traitement par ciprofloxacine ou gentamicine, et 35% pour la doxycycline. Les causes de ces échecs ne sont pas bien élucidées à l’heure actuelle. L’analyse de la littérature ainsi que la détermination de la sensibilité de 59 souches françaises de F. tularensis subsp. holarctica à 18 antibiotiques, confirment qu’aucune souche isolée à ce jour ne présente de résistance acquise à ces trois familles d’antibiotiques, qui représentent le traitement de première ligne de la tularémie. Les fluoroquinolones (en particulier la ciprofloxacine et la lévofloxacine) présentent concentrations minimales inhibitrices les plus basses, devant la gentamicine et la doxycycline. Les données disponibles in vitro et en modèle animal étant corrélées aux données humaines en termes d’efficacité et de taux d’échecs thérapeutiques, il semble néanmoins préférable de positionner la ciprofloxacine en première ligne pour le traitement des formes modérées de tularémie et de limiter l’utilisation de la doxycycline aux cas de contre-indication aux fluoroquinolones. L’azithromycine et la télithromycine ont été identifiées comme des alternatives thérapeutiques envisageables en cas d’infection par une souche de biovar I de F. tularensis subsp. holarctica lorsqu’existe une contre-indication aux traitements de première ligne. Des études en modèles animaux restent néanmoins nécessaires pour conforter ces dernières observations. La sélection in vitro de souches résistantes aux fluoroquinolones est possible, ce qui suggère la possibilité d’émergence de mutants résistants in vivo pour expliquer les taux d’échec thérapeutiques. Les principales mutations de résistance aux fluoroquinolones chez F. tularensis sont observées au niveau des gènes gyrA et gyrB codant pour les topoisomérases de type II. L’impact fonctionnel de mutations de résistances aux fluoroquinolones a été caractérisé in vitro chez F. novicida, pris comme modèle de bactérie avirulente proche de F. tularensis. L’activité de superenroulement et de clivage de l’ADN en présence de fluoroquinolones a été déterminée suite à la reconstruction in vitro de complexes GyrA/GyrB fonctionnels. La résistance aux fluoroquinolones était la plus forte en cas de mutation D87G/D87Y pour la sous-unité GyrA ou +P466 pour la sous-unité GyrB. La mutation P43H située en dehors du QRDR de GyrA est à l’origine d’un plus faible niveau de résistance. La mutation D487R-∆K488 en dehors du QRDR de GyrB ne confère pas de résistance intrinsèque mais potentialise l’effet d’une mutation D87G concomitante. En revanche, l’identification de mutations de résistance in vivo au sein des QRDR des gènes gyrA et gyrB chez des patients en situation d’échec thérapeutique traités par une fluoroquinolone est demeurée négative. Enfin, notre recherche a permis d’identifier de nouveaux composés de synthèse de structure bis-indolique possédant des activités antibactériennes. Ces composés sont bactériostatiques vis-à-vis de F. tularensis mais bactéricides vis-à-vis des staphylocoques y compris vis-à-vis de souches multi-résistantes de Staphylococcus aureus avec des CMI90 évaluées à 2mg/L chez F. tularensis et S. aureus pour le composé le plus actif. La faible solubilité de ces composés en milieu aqueux, leur forte liaison aux protéines plasmatiques ainsi que la recherche de leur mécanisme d’action original appellent néanmoins de nombreux développements futurs. / Tularemia is a zoonosis caused by the highly pathogenic bacterium Francisella tularensis. The most virulent subspecies, F. tularensis subsp. tularensis, is found only in North America while the subspecies F. tularensis subsp. holarctica is present in the whole Northern hemisphere. In France, all strains belong to the biovar I of the subspecies holarctica and more specifically to the phylogenetic subclade B.FTNF002-00. Although tularemia is usually not a severe disease in France, many patients suffer from therapeutic failures despite receiving an appropriate treatment. These treatments failures are observed in up to 25% of patients treated with ciprofloxacin or gentamicin, and up to 35% if patients treated with doxycycline. The causes of those therapeutic failures remain poorly elucidated. Analysis of the literature and determination of the susceptibility of 59 French F. tularensis subsp. holarctica strains to 18 antibiotics confirmed that to date, no strain with acquired resistance to any of the first-line antibiotics used for treatment of tularemia have been isolated. The fluoroquinolones (in particular ciprofloxacin and levofloxacin) exhibit the lowest minimal inhibitory concentrations, compared to gentamicin and doxycycline. Data obtained in vitro and in animal models are concordant with human data concerning the efficacy of antibiotics and therapeutic failure rates. Thus, we advocate the use of ciprofloxacin as first-line treatment for mild form of tularemia, and the use of doxycyclin only as a second-line treatment in patients with contraindications to fluoroquinolones. Azithromycin and telithromycin may also be considered as potential therapeutic alternatives for tularemia cases caused by biovar I strains of the susbspecies holarctica, but only for patients with contraindications to first-line antibiotics. Further data in animal models are however required to consolidate our in vitro data. The in vitro selection of fluoroquinolone-resistant strains of F. tularensis has been reported. This suggests that the in vivo selection of such resistant mutants may occur. In vitro, the main fluoroquinolone resistance mutations occur in the gyrA and gyrB genes that encode type II topoisomerases of F. tularensis. We have characterized the functional impact of such mutations in avirulent F. novicida strains, taken as a surrogate of F. tularensis. Supercoiling and DNA cleavage activity of GyrA/GyrB complexes reconstituted in vitro have been determined in the presence of fluoroquinolones. Fluoroquinolone resistance level was the highest in strains with a D87G/D87Y mutation in the GyrA subunit or +P466 mutation in the GyrB subunit. The mutation P43H located outside the GyrA Quinolone-Resistance-Determining-Region (QRDR) confered significant but lower fluoroquinolone resistance. The mutation D487R-∆K488 also outside GyrB QRDR did not cause fluoroquinolone resistance by itself, but increased the resistance level in case of concomitant D87G mutation. No mutation could be identified in vivo in the QRDR of gyrA and gyrB genes amplified from clinical samples collected in patients treated with a fluoroquinolone, although some of them experienced therapeutic failure. Finally, while searching for new antibiotic compounds, we identified new synthetic bis-indolic derivatives with antibacterial activity. Lead compounds were only bacteriostatic against F. tularensis but bactericidal against staphylococci including against multi-drug-resistant Staphylococcus aureus. MIC90 were measured at 2mg/L for F. tularensis and S. aureus strains for the most active compound. However, many developments are still required to improve their solubility in water, decrease their plasma proteins binding and elucidate their original mechanism of action.

La dépression et les troubles anxieux chroniques constituent une problématique majeure de santé publique et les stratégies pharmacologiques conventionnelles que sont les antidépresseurs apparaissent à la fois souvent d'une inefficacité limitée et potentiellement source d?iatrogénie. Pour mettre au point de nouveaux traitements la modélisation animale demeure une solution de choix pour évaluer les modifications comportementales induites par ces nouvelles molécules. La création de nouvelles modélisations plus performantes apparaît comme un champ d'investigation important afin d'optimiser le rôle de filtre de la recherche préclinique. L'objectif du travail que nous présentons et de proposer des traitements exploitant des nouvelles voies pharmacologiques comme celles interagissant avec les voies opioïdergiques ou présentant des propriétés anti-oxydantes. Les résultats présentés mettent ainsi en lumière à travers le test de la nage forcée chez le rat, les effets antidépresseurs d'un nouvel inhibiteur des enképhalinases, l'opiorphine, et d'un extrait polyphénolique de cacao. Nous présentons secondairement le développement de nouvelles modélisations animales chez le rat afin de mieux appréhender deux types de situations cliniques. Nous proposons d?une part une nouvelle modélisation éthologique de l'anxiété extrême dans laquelle plusieurs traitements de référence (fluoxétine en chronique et en aiguë, imipramine, diazépam et clonazépam) révèlent une efficacité comparable à celle observée en clinique dans le cadre du trouble panique. D'autre part, une modélisation associant une procédure de stress chronique modérée à une carence chronique en donneurs de méthyle (folates notamment) conduit à manifestations comportementales et des modifications neurobiochimiques originales dont le rapprochement avec certaines psychopathologies peut être discuté (dépression mélancolique et trouble panique). Les données que nous présentons sur l'évaluation de nouveaux traitements comme sur la mise au point de nouvelles modélisations posent des bases prometteuses pour la recherche sur les troubles anxio-dépressifs / Depression and chronic anxiety disorders are a major public health problem and conventional pharmacological strategies (antidepressants) appear often ineffective with a iatrogenic potential. In order to develop new treatments, animal models remain a solution of choice for evaluating the behavioral changes induced by these molecules. Creating new models more efficient seems to be a major field of investigation in order to optimize the role of filter of preclinical research. The objective of the work we present here is to propose some treatments exploiting new pharmacological tools like those with interaction with opioidergic pathways or with anti-oxidant properties. The results in the forced swimming test in rats show the antidepressant effects of a novel enkephalinase inhibitor, opiorphin, and of a cocoa polyphenolic extract. Secondarily, we present the development of new animal models in rats to better understand two types of clinical situations. We propose a new ethological model of extreme anxiety in which several reference treatments (chronic and acute fluoxetine, imipramine, diazepam and clonazepam) showed an efficacy comparable to that observed in panic disorder. On the other hand, a modeling procedure combining chronic mild stress to a chronic deficiency of methyl donors (folates in particular) leads to behavioral manifestations and neurobiochemical changes whose similarity with certain psychopathologies can be discussed (melancholic depression and panic disorder). The evaluation of new treatments and the development of new models set the foundations for promising research on anxiety-depressive disorders

Depression och ångestsyndrom hör till de vanligaste psykiska sjukdomarna och medför stora aktivitetsbegränsningar inom arbete, fritid och personlig vård. Trots det finns det få litteraturstudier om arbetsterapeutiska interventioner för denna målgrupp. Syftet var att beskriva arbetsterapeutiska interventioner för vuxna personer med depression eller ångestsyndrom. En systematisk litteraturstudie genomfördes i Pubmed, PsycInfo, Scopus och Cinahl. Arton artiklar inkluderades. Artiklarna analyserades med stöd av Friberg (2012). Tre huvudteman identifierades: 1) Att hjälpa klienterna få ökad insikt om sin livssituation, 2) Att lära klienten hantera och förebygga symtom och 3) Aktivitet som medel i interventionen. Resultatet har betydelse för hur arbetsterapeuter kan välja, kommunicera och genomföra interventioner samt för framtida forskning. Betydelsen av val av behandlingsstrategier i interventionerna diskuterades också. / Depression and anxiety disorders are among the most common mental disorders and lead to serious activity limitations in work, leisure and self-care.  In spite of this, there are few systematic reviews on occupational therapy interventions for this client group. The study aim was to describe occupational therapy interventions for adults with depression or anxiety disorders. A systematic review was conducted in Pubmed, PsycInfo, Scopus and Cinahl. Eighteen articles were included. The articles were analyzed in accordance with Friberg (2012). Three main themes were identified: 1) To help clients gain insight into their life situation, 2) To teach clients how to manage and prevent symptoms, and 3) Occupation as means in the intervention. The findings have implications for how therapists can choose, communicate and carry out interventions as well as for future research. The importance of therapeutic strategies in the interventions was also discussed.

O glioblastoma (GBM) é um tumor extremamente agressivo e resistente aos tratamentos convencionais. Os agentes utilizados na quimio- e radioterapia são indutores de danos no DNA, por induzirem quebras de fita simples (SSBs) e quebras de fita dupla (DSBs), as quais são letais para as células, mas quando eficientemente reparadas pelas células tumorais tornam estas resistentes. As principais vias de reparo de DSBs são: a via por recombinação homológa (Homologous Recombination HR) e por recombinação não- homóloga (Non-homologous end joining NHEJ). As proteínas de reparo participantes dessas vias têm sido estudadas como potenciais alvos moleculares na terapia contra o câncer. A estratégia empregada no presente trabalho foi a de inibir a via de reparo HR em células já comprometidas para a via NHEJ. Para isso foi utilizado o inibidor de RAD51 (uma das principais proteínas da via HR), 3-chloro-1-(3,4-dichlorophenyl)-4- morpholinylo-1H-pyrrole-2,5-dione, conhecido como RI-1(Calbiochem), testado em células de glioma M059J e M059K (deficientes e proficientes para DNA-PK, respectivamente), irradiadas com raios-X. Diferentes ensaios foram realizados para o teste com o inibidor RI-1 em células irradiadas ou não e comparados ao controle sem tratamento. Os resultados dos ensaios de sobrevivência clonogênica mostraram que a concentração de 40 M do inibidor RI-1 exerceu um maior efeito inibitório sobre a capacidade de as células se dividirem e formarem colônias. O RI-1 induziu alterações significativas na cinética do ciclo celular predominantemente na linhagem selvagem M059K, nos tempos de 24 e 72 h. Apesar de a linhagem M059J não mostrar alterações significativas na cinética do ciclo celular, esta demonstrou sensibilidade à irradiação, conforme demonstrado pela cinética de reparo das DSBs, sendo mais lenta em relação à M059K, demonstrando o comprometimento do reparo NHEJ na linhagem mutante para DNA-PK. A expressão das proteínas LIG3, XRCC1 e PARP-1 foram analisadas no tempo de 15 minutos e 24 h após a irradiação. Na presença do inibidor RI-1, a expressão da LIG3 foi aumentada em células M059K (15 min e 24 h) comparadas ao grupo controle. Já a linhagem M059J apresentou uma elevada expressão das proteínas XRCC1 e PARP-1 apenas em 15 min em relação ao controle; esses dados indicaram que um possível reparo de DSBs envolvendo essas proteínas pode ter sido ativado logo nos primeiros minutos após a indução de danos nessas células. O conjunto dos resultados deste trabalho sugere um papel atuante do inibidor RI-1 em células comprometidas para o reparo NHEJ, isto é, a linhagem M059J, levando à sugestão de que vias alternativas de reparo podem possivelmente estar envolvidas na resistência das células tumorais aos tratamentos convencionais. / Glioblastoma (GBM) is an extremely aggressive and resistant tumor to conventional treatments. The agents used in chemotherapy and radiotherapy are inducers of DNA damage, since they induce single strand breaks (SSBs) and doublestrand breaks (DSBs), which are lethal to cells, but when efficiently repaired by tumor cells make them resistant to antitumoral agents. The main repair pathways for DSBs are the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Proteins participating in these processes have been studied as potential molecular targets in cancer therapy. Thus, the strategy employed in this work involved the inhibition of HR repair pathway in cells already committed to the NHEJ pathway, aiming to sensitize irradiated GBM cells. An inhibitor of RAD51 (one of the major HR proteins) was used: 3-chloro-1-(3,4-dichlorophenyl) -4 morpholinylo-1Hpyrrole- 2,5-dione, known as RI-1 (Calbiochem); this compound was tested in GBM cells, M059K and M059J (proficient and deficient for the DNA-PK, respectively) irradiated with X-rays. Various assays were performed to test the inhibitory property of RI-1 in irradiated cells and the combination of the inhibitor with X-irradiation, compared with the untreated control. The results of clonogenic survival showed that 40 M of RI-1 inhibitor exerted a higher inhibitory effect on the ability of cells to divide and form colonies. The RI-1 induced changes in cell cycle kinetics predominantly in the wild-type M059K, at 24 and 72 h. Although M059J did not show significant changes in cell cycle kinetics, these cells showed sensitivity to X-irradiation, as shown by the kinetics of DSB repair (gamma-H2AX foci), which was slower compared to M059K, demonstrating the commitment of the NHEJ repair in M059J (mutant for DNA-PK). The expression of LIG3, PARP-1 and XRCC1 proteins were analyzed at 15 min. and 24 h after irradiation. In the presence of the inhibitor RI-1, LIG 3 expression was increased in M059K cells (15 min. and 24 h) compared to the control group. M059J cells showed a high expression of XRCC1 and PARP-1 only at 15 min., compared to the control. These data indicated that a possible repair of DSBs involving these proteins may have been activated in the first minutes after DNA damage induction. The overall results of this study suggest that RI-1 inhibitor was efficient to influence cellular responses in cells committed to the NHEJ repair, i.e. M059J cell line, leading to the hypothesis that alternative repair pathways may be possibly involved in the resistance of tumor cells.

O glioblastoma (GBM) é um tumor extremamente agressivo e resistente aos tratamentos convencionais. Os agentes utilizados na quimio- e radioterapia são indutores de danos no DNA, por induzirem quebras de fita simples (SSBs) e quebras de fita dupla (DSBs), as quais são letais para as células, mas quando eficientemente reparadas pelas células tumorais tornam estas resistentes. As principais vias de reparo de DSBs são: a via por recombinação homológa (Homologous Recombination HR) e por recombinação não- homóloga (Non-homologous end joining NHEJ). As proteínas de reparo participantes dessas vias têm sido estudadas como potenciais alvos moleculares na terapia contra o câncer. A estratégia empregada no presente trabalho foi a de inibir a via de reparo HR em células já comprometidas para a via NHEJ. Para isso foi utilizado o inibidor de RAD51 (uma das principais proteínas da via HR), 3-chloro-1-(3,4-dichlorophenyl)-4- morpholinylo-1H-pyrrole-2,5-dione, conhecido como RI-1(Calbiochem), testado em células de glioma M059J e M059K (deficientes e proficientes para DNA-PK, respectivamente), irradiadas com raios-X. Diferentes ensaios foram realizados para o teste com o inibidor RI-1 em células irradiadas ou não e comparados ao controle sem tratamento. Os resultados dos ensaios de sobrevivência clonogênica mostraram que a concentração de 40 M do inibidor RI-1 exerceu um maior efeito inibitório sobre a capacidade de as células se dividirem e formarem colônias. O RI-1 induziu alterações significativas na cinética do ciclo celular predominantemente na linhagem selvagem M059K, nos tempos de 24 e 72 h. Apesar de a linhagem M059J não mostrar alterações significativas na cinética do ciclo celular, esta demonstrou sensibilidade à irradiação, conforme demonstrado pela cinética de reparo das DSBs, sendo mais lenta em relação à M059K, demonstrando o comprometimento do reparo NHEJ na linhagem mutante para DNA-PK. A expressão das proteínas LIG3, XRCC1 e PARP-1 foram analisadas no tempo de 15 minutos e 24 h após a irradiação. Na presença do inibidor RI-1, a expressão da LIG3 foi aumentada em células M059K (15 min e 24 h) comparadas ao grupo controle. Já a linhagem M059J apresentou uma elevada expressão das proteínas XRCC1 e PARP-1 apenas em 15 min em relação ao controle; esses dados indicaram que um possível reparo de DSBs envolvendo essas proteínas pode ter sido ativado logo nos primeiros minutos após a indução de danos nessas células. O conjunto dos resultados deste trabalho sugere um papel atuante do inibidor RI-1 em células comprometidas para o reparo NHEJ, isto é, a linhagem M059J, levando à sugestão de que vias alternativas de reparo podem possivelmente estar envolvidas na resistência das células tumorais aos tratamentos convencionais. / Glioblastoma (GBM) is an extremely aggressive and resistant tumor to conventional treatments. The agents used in chemotherapy and radiotherapy are inducers of DNA damage, since they induce single strand breaks (SSBs) and doublestrand breaks (DSBs), which are lethal to cells, but when efficiently repaired by tumor cells make them resistant to antitumoral agents. The main repair pathways for DSBs are the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Proteins participating in these processes have been studied as potential molecular targets in cancer therapy. Thus, the strategy employed in this work involved the inhibition of HR repair pathway in cells already committed to the NHEJ pathway, aiming to sensitize irradiated GBM cells. An inhibitor of RAD51 (one of the major HR proteins) was used: 3-chloro-1-(3,4-dichlorophenyl) -4 morpholinylo-1Hpyrrole- 2,5-dione, known as RI-1 (Calbiochem); this compound was tested in GBM cells, M059K and M059J (proficient and deficient for the DNA-PK, respectively) irradiated with X-rays. Various assays were performed to test the inhibitory property of RI-1 in irradiated cells and the combination of the inhibitor with X-irradiation, compared with the untreated control. The results of clonogenic survival showed that 40 M of RI-1 inhibitor exerted a higher inhibitory effect on the ability of cells to divide and form colonies. The RI-1 induced changes in cell cycle kinetics predominantly in the wild-type M059K, at 24 and 72 h. Although M059J did not show significant changes in cell cycle kinetics, these cells showed sensitivity to X-irradiation, as shown by the kinetics of DSB repair (gamma-H2AX foci), which was slower compared to M059K, demonstrating the commitment of the NHEJ repair in M059J (mutant for DNA-PK). The expression of LIG3, PARP-1 and XRCC1 proteins were analyzed at 15 min. and 24 h after irradiation. In the presence of the inhibitor RI-1, LIG 3 expression was increased in M059K cells (15 min. and 24 h) compared to the control group. M059J cells showed a high expression of XRCC1 and PARP-1 only at 15 min., compared to the control. These data indicated that a possible repair of DSBs involving these proteins may have been activated in the first minutes after DNA damage induction. The overall results of this study suggest that RI-1 inhibitor was efficient to influence cellular responses in cells committed to the NHEJ repair, i.e. M059J cell line, leading to the hypothesis that alternative repair pathways may be possibly involved in the resistance of tumor cells.

Studies indicate that, except for anger, many men tend to avoid expressing their feelings, especially those feelings indicative of personal vulnerability and emotional dependency (Levant, Hall, Williams, & Hasan, 2009). Men are frequently portrayed as lacking the ability to recognise, own and find words with which to express their feeling experiences; this is captured by the term alexithymia (Levant, Hall, Williams, & Hasan, 2009). Defined by ‘restrictive emotionality’, alexithymia literally indicates ‘without words for emotions’. Roland Levant has contended that men who are strongly influenced by ideas of traditional masculinity tend to be alexithymic (Levant, Hall, Williams, & Hasan, 2009). The central aim of this study was to focus on and understand the emotional experiences of participants of all-male or gender-homogenous group psychotherapy of this study. The study used a qualitative approach to understand how men emotionally engage or do not engage and express their emotions. Men who had been in all-male group psychotherapy were purposively selected to participate in this study. In-depth interviews guided by a semi-structured questionnaire were conducted and analysed according to the thematic analytic method. This study explored and described the accounts of lived emotional engagements of male participants in an all-male psychotherapy group. The study sought to highlight the significance of an explicit masculine framework with male emotions within a framework of non-deficit assumptions. The non-deficit approach to men privileges the strengths and unique contributions that men make as partners and fathers (Dollahite & Hawkins, 1998). This study explored and described the accounts of lived emotional engagements of male participants in an all-male psychotherapy group. The study sought to highlight the significance of an explicit masculine framework with male emotions within a framework of non-deficit assumptions. The non-deficit approach to men privileges the strengths and unique contributions that men make as partners and fathers (Dollahite & Hawkins, 1998). This study aspired to demonstrate that a gender-conscious model in working with male emotions enhances men’s capacity for a quality and depth of emotional engagement that echoes the more optimistic research on the male capacity for self-reflection and openness to subjective transformation (Kiselica, 2003). / Psychology / Ph. D. (Psychology)